1. Field of Invention
The present invention relates generally to methods and apparatus for performing biochemical and biomedical assays, and, more particularly, to methods and apparatus for measuring reagent volumes in such assays. In one aspect, the present invention includes a method and apparatus for determining the volumes of reagents used in automated biochemical and biomedical assay apparatus.
2. Background
Affinity binding assays are often used to detect the presence of a molecule associated with a disease condition or biological state. These assays often are based upon "binding pairs", i.e., a complementary pair of molecules which exhibit mutual affinity or binding capacity. Typically, one of the molecules of the binding pair is designated the "ligand" while the other molecule of the binding pair is designated the "antiligand." The ligand is generally considered to be a donor, whereas the antiligand may be a receptor, analyte, or target. The designation of a ligand and an antiligand is arbitrary in that the designation is dependent upon which molecule is to be detected. The binding pair may comprise two complementary nucleic acids, antigens and antibodies, drugs and drug receptor sites, and enzymes and enzyme substrates.
Typically, one member of the biological binding pair, e.g., the ligand, is immobilized on a solid support surface such as plastic, glass, or nitrocellulose paper. Methods used to immobilize immunological agents, peptides, and nucleic acids on a solid support are well known in the art. Nucleic acid sequences that are specific for particular disease states, as well as immunological agents such as antibodies that are specific for a particular disease state, are commonly reported in the scientific press and various other published applications. As such, an appropriate ligand to use for a given antiligand is readily determined.
A sample, which potentially contains the molecule of interest, i.e., the molecule to be detected, is applied to the ligand-containing solid support surface. In general, the sample is a fluid sample. The fluid sample and the support are then incubated in order to provide an opportunity for the molecule to be detected to bind to the immobilized ligand. During this period of incubation, the fluid sample and the support surface may be agitated to facilitate the flow of the fluid sample over the support surface to maximize the opportunity for the target molecule, or the molecule to be detected, to be received by the immobilized ligand. After this period of incubation, the unbound fluid sample is removed. The target molecule, if present, forms a complex with the immobilized ligand.
After the unbound fluid sample is removed, additional reagents which are capable of reacting with the complex, or the target captured by the immobilized ligand, are applied to the support surface. These reagents typically include another, i.e., a second, ligand which is capable of binding to the complex or the target. This second ligand has a label, or a molecular section which may be detected. Typical labels include, but are not limited to, radioactive isotopes, enzymes, luminescent agents, precipitating agents, and dyes.
The support surface is monitored for the presence of an indication that a target molecule is present. By way or example, the presence of a particular color on the support surface may indicate that a target molecule is present. In some cases, the support surface may be monitored with fluorescent light in order to detect the presence of a target molecule on the support surface. The presence, or absence, of the appropriate indication is then correlated with the disease condition or biological state of the sample source. For example, where the ligand employed in an assay is an antibody directed to human immunodeficiency virus (HIV), the presence of an indicator implies the presence of anti-HIV antibodies in the sample fluid--an indication that the sample donor is infected with HIV.
Automated affinity binding assay processes are generally preferred over non-automated processes, as automated processes are more efficient and easier to control with less chance for random procedural errors. Automated affinity binding assay processes generally provide for the accurate and precise delivery of assay reagents and other necessary fluids to individual reaction vessels which hold the test strips used in affinity assay processes. One automated affinity binding assay process utilizes the Chiron RIBA.TM. Processor System which is commercially available from Chiron Corporation of Emeryville, Calif.
Recently, the FDA has provided guidelines relating to the verification of the volumes of assay reagents dispensed into reaction vessels used in automated affinity assays. By way of example, if an incorrect volume of an assay reagent is used in an affinity assay, the results of the affinity assay may be inaccurate, and, in a worst-case scenario, an individual afflicted with a deadly condition may be falsely diagnosed as being healthy. Clearly, therefore, assuring the accuracy of affinity assays is a critical concern for those in the medical diagnostic arts. Along these lines, the FDA has provided guidelines which suggest that volume measurements taken in reaction vessels for automated assay devices vary from the actual reagent volume by no more than ten percent.
In general, the volumes of assay reagents dispensed into a reaction vessel are monitored using "contact" methods, such as with an electro-chemical probe. For example, a two-pronged probe is placed into contact with an assay reagent in a reaction vessel, and the resistance between the two prongs of the probe is measured in order to determine the volume of assay reagent in the reaction vessel. These methods, however, are invasive and can lead to contamination problems if the probes used in the measuring process are not properly cleaned after each measurement. By way of example, for affinity tests involving the use of polymerase chain reaction (PCR), accidentally contamination of one sample with even minute amounts of DNA from another sample may lead to spurious analytical results, which may have any number of unforeseen consequences for the sample's donor.
Furthermore, the performance of affinity assays has been limited by processing inefficiencies associated with the need to insert probes into assay reagents, take readings, remove the probes, and clean the probes. The performance issues, together with the contamination concerns, make conventional methods for automated assay reagent volume dispense verification less than desirable.
Therefore, what is desired are efficient, non-invasive, i.e., non-contact, methods and apparatus for automated assay reagent volume dispense verification. Still more desirable are methods and apparatus that provide automated assay reagent volume verification that conform with FDA guidelines.